The objectives of the proposed research are the following: (1) To characterize in detail the different levels of the subunit structure of Lumbricus hemoglobin. This will include determination of the molecular masses and number of copies of the constituent polypeptide chains and of their arrangement in the subunits making up the native molecule. (2) To compare the subunit structure of Lumbricus hemoglobin with the subunit structure of other annelid hemoglobins, namely those of Arenicola Nephtys and Tubifex. (3) To investigate the reassociation of the different hemoglobins subsequent to dissociation at alkaline pH, including the role of divalent cations. (4) To determine the distribution of heme among the subunits of the native hemoglobins and the role of heme-carrying subunits in the function of these hemoglobins. (5) To determine the location of Cu and Zn binding sites among the subunits of the foregoing hemoglobins and correlate their presence with functions other than the binding of oxygen. The subunit structure of hemoglobins will be determined using gel filtration and polyacrylamide gel electrophoresis in the presence of detergents and at alkaline pH in combination with ultracentrifugation, electron microscopy and low angle x-ray and neutron scattering. The number of copies of the isolated subunits will be determined by quantitative polyacrylamide gel electrophoresis. Identification of the constituent polypeptide chains will be completed using end group analysis together with stepwise Edman degradation whenever necessary. The reassociation of the hemoglobins will be investigated by gel filtration and the various products characterized by electron microscopy and low angle x-ray and neutron scattering. The function of the heme-containing subunits will be studied by measurement of oxygenation of the individual subunits and reconstituted aggregates thereof in comparison with that of the native molecule. The determination of Cu and Zn in the subunits of the hemoglobin will be carried out using x-ray fluorescence spectrometry.